Reagents and tests for syphilis

ABSTRACT

An antigen preparation for use in the detection of syphilis and other treponemal diseases comprising an antigen reagent and a class of dyes and a method of using said antigen preparation, are disclosed.

Umted States Patent 11 1 1111 3,882,224 Forgione 1 1 May 6, 1975 I541 REAGENTS AND TESTS FOR SYPHILIS 2,301,717 11/1942 Terry 424/11 770, 72 II 956 El .,441l salvam" Stammfli 530 253 1;:963 Brit/ er 454/12 Cum 3,666,421 5/1972 Price 424 12 x Assignccz American Cyanamid p y, 3,689,632 9/1972 Mlzushlmu .t 424/12 Stumfnrd, Conn OTHER PUBLICATIONS 22 FHcd: Sept. I]! 973 Berger, Chem. AbS., VOI. 38, 1944, pp. 3996-3998,

Berger, BriL Exptl. Path., V01 24, 1943, pp. 252-261. I211 App]. No; 396,l69

Primary ExaminerAlbcrl T. Meyers 152 u.s. c1. 424/8; 424/3; 424/7, Hammer-A Fagclw" 424 424/ Attorney, Agent, or Firm-Frz1nk M. Van Rict [51] Int. Cl. (10in 3l/02; G01n 31/22; ()(Hn 33/16 1581 Field of Search 424/3, 7,11, 11, 12, 13 1571 ABSTRACT An anligcn preparation for use in the detection of [56] References Cited syphilis and other treponcmal diseases comprising an n- STATES PATENTS antigen reagent and a class of dyes and a method of 2 H] 976 3H9 hughlin 424; using said antigen preparation, are disclosed. 2,112,496 3/1938 Ide 2 424/8 9 Claims, N0 Drawings 3,882,224 1 2 REAGENTS AND TESTS FOR SYPHlLlS SUMMARY OF THE INVENTION l have now found a novel visual method for the detec- BACKGROUND OF THE IN ENHON tion of syphilis which has material advantages over The use ofserological tests for the detection of syphiother techniques such as the use of charcoal or latex lis and other treponemal diseases has become more and emulsions. Since my system does not utilize solid partimore commonly practiced in recent years. These tests cles. no light scattering is caused and therefore a more are usually based on an agglutination reaction and are easily visible particle is produced ifa positive serum or conducted in clinics and doctors offices preparatory to plasma is used, Therefore. the instant test is more accumore extensive diagnosis of the patient. rate and reliable.

The most commonly practiced test constitutes the 10 According to my novel test. when my novel antigen use of a finely divided solid such as charcoal in conreagent is contacted with an antibody-containing Serum junction with a card having a surface color contrasting or lasma. a complex is formed which forms a coaccerto that of the solid material. see US. Pat. No. vate with one component ofthe reagent. A positive test 3.074.853. In practice. the test utilizes a common antigen liquid which is buffered and to which is added the charcoal. A drop of the resultant antigen solutioncharcoal suspension is thcn placed on a test card in admixture with one drop of serum. The card is then shaken and the results are visually interpreted. Addis is indicated by colored flocs which are especially noticeable on a light background, e.g.. a white card.

DESCRIPTION OF THE INVENTION INCLUDING PREFERRED EMBODIMENTS As mentioned briefly above. I have discovered a 2U ttonal (.IIIJgIlUSIIC reagents I'ILIVC 'tIISU I'CCCI'IIIY been p21 novel antigen preparation for use n arrynyg ul an age [Emmi See Pill- 3564389 and 1 4 glutination test for syphilis and other treponemal dis- WhilL Y Brit l @1161 Dist l VOL eases. The antigen preparation consists of two basic in pages 290-294 sets out a modified Laughlin floccula gradients th fir t being a common V.D.R.L.. U.S.R. tion test using a Kahn antigen coupled with a scarlet or Reiter antigen reagent. known to those skilled in the red stain and a Wassermann complement-fixation techart and more completely identified in US. Pat. No. nique. using a cardiolysin antigen. 3.564.089. mentioned above.

The known test methods. while resulting in adequate The second component of my novel antigen preparasyphilis detection in most cases. are by no means pertion is a dye compound having the formula wherein AR is 2 R1 or it feel and each possesses its own difficulties. The chain 5 wherein R and R are. individually. hydrogen. lower coal test. for example, is very difficult to interpret due alkoxy. lower alkyl, amino, monoalkylamino. dialkylto the tendency of the positive and negative results to amino or monoarylamino groups. no more than one of be similar in many instances. Where a definitely posi- R and R being hydrogen and R is hydrogen, amino or tive test is encountered. no difficulty arises. however. monoarylamino groups. R is an SO Na group. Z is SO in cases where the results are in doubt. further extenso or Cl. X is oxygen. sulfur orN wherein Y is or lower hydrogen 1 alkyl gr up R5 sive tests must be conducted before a definite conclu R and R are. individually. hydrogen. lower alkoxy or sion can be drawn. With regard to the scarlet red stain lower alkyl groups and AR is test, the results tend to be nebulous because ofthe faint pink color ofthe aggregated particles and the concomitant difficulty in analyzing and interpreting the test. 65

It has also been well recognized that other commonly 7 or 9 used tests have been known to give completely erroneous results in 2.0 percent ofthe tests run and false posi- 8 tivc results in 20 percent of the test cases. R

wherein R is hydrogen or lower alkyl. R and R are. individually. hydrogen. amino. monoarylamino. lower alkyl. dialkylamino. monoalkylamino. hydroxy or nitro groups. no more than one of R and R being hydrogen and R and R" are. individually. hydrogen. amino. monoarylamino. monodialkylaminoarylamino, or hydroxy groups. and the zinc double chlorides thereof.

The dye compounds and mixtures are present in my novel antigen preparations in conjunction with solvents, buffers. etc.. in a manner as is known in the art. These solvents. buffers etc. per se form no part of the instant invention and are used as described in known antigen systems such as those discussed in the aboveidentified U.S. patents. hereby incorporated herein by reference.

The components of the novel antigen preparation of my invention are employed in concentrations ranging from about 0.1 percent to about 25.0 percent. by weight. of the standard V.D,R.L.. U.S.R. or Reiter antigen and from about 0.000l percent to about 0.2 percent. by weight, of the dye compounds. said weights being based on the total weight of the resultant mixture including solvents. e.g.. water. alcohol. etc.. and buffer.

By "standard V.D.R.L. and U.S.R. antigen. as used herein. is meant the compositions set forth and prescribed by the Manual of Tests for Syphilis (i969). U.S. Dept. of Health. Education and Welfare. Public Health Service. National Communicable Disease Center.

A third component may also be present in my novel antigen preparations. This component is preferred but not critical and comprises a flocculating agent. The flocculating agent can be employed in amounts ranging from about 0.000l to about 0.05 percent, by weight, based on the total weight of the novel antigen preparation of the instant invention, i.e.. standard antigen and dye compound. and includes such known flocculants as vinylminidazoline. acrylamide. acrylic acids, acrylonitrile. styrene. maleic anhydride, etc.. polymers and copolymers thereof with each other and other known copolymerizable monomers.

In practice. the flocculating agent aids in the precipitation and coaccervation of the antigen-antibody complex and thereby aids in the visual detection of the positive test.

In preparing the antigen formulations of the present invention. standard V.D.R.L. antigen can be used. The dye compound may be incorporated into an alcoholic solution of the V.D.R.L. antigen or added after the rea gent antigen has been prepared in buffer. lfa flocculating agent is to be incorporated into the basic preparation. it is also added with slight agitation at this time.

The U.S.R. antigen-dye compound formulation is made by first preparing the \/.D.R.L. antigen suspension in buffer. This system is then centrifuged in a stainless steel vessel at approximately 2.000 g for 15 minutes. The fluid is decanted and the sides of the vessel wiped dry. The sediment is suspended in U.S.R. sus pending medium which consists ofa known and appropriate mixture of monopotassium phosphate. disodium phosphate. merthiolate. choline chloride and ethylenedinitrilotetraacetic acid. The dye compound or compounds may be incorporated into the alcoholic solution of \".D.R.L. antigen or the U.S.R. antigen after its suspension of U.S.R. suspending medium.

When standard Reiter antigen is used. the dye compound is merely added thereto and the resultant antigen preparation is used as such in the test.

The tests utilizing the antigen preparations of the instant invention entail the use of a test card which may or may not have a flocculating agent deposited thereon. The test card should be smooth in order to render the test results easily discernible. Although it has been previously indicated in the prior art that the surface must be wettable. I have found that such is not the case and a wettable surface is merely preferred. The card may be composed of well calendered paper or cardboard and 10 may be absorbable. however. only small degrees of ab sorbability are preferred. One feature of the use of my novel antigen preparations is that the test may be read for the test card whether the spot is wet or dry and therefore results can be ascertained more rapidly than when using other techniques. The card may also be a laminate of a paper or cardboard base having a waterpermeable or water-impermeable material coated thereon such as polyethylene. The paper itself or the coating. however. should be of a color which is con trasting with regard to the color of the dye employed in the test. Basically. any card similar in construction and composition to that disclosed in US Pat. No. 3.074.853 may be used. The flocculating agent may be deposited on the card. if desired. by merely evaporating it from a solution after having placed a solution thereof thereon. The solvent may be either water or an organic material such as methylene chloride. etc. An adhesive may also be added to the card beforehand to insure adequate adhesion of the flocculating agent thereto. A

suitable adhesive for this purpose is methyl cellulose.

The same flocculating agents as mentioned above with regard to direct addition thereof to the antigen preparation may be used. The flocculating agent is usually deposited in selected areas only of the test card. these areas usually being designated as a circle. etc.. the area within which the test is actually conducted. When the flocculating agent is present on the card per se. it is not necessary to add further flocculating agent to the antigen preparation being used to conduct the test.

Tests are usually carried out by shaking the cards after the antigen preparation and serum have been added dropwise thereto. The shaking may be carried out on. for example. a horizontal disc about 10 inches square at about 80-240 strokes per minute with a slight 45 eccentric motion. However. hand shaking is also effectwo.

The following examples are set forth for purposes of illustration only and are not to be construed as limitations on the instant invention except as set forth in the appended claims. All parts and percentages are by weight unless otherwise indicated.

EXAMPLE 1 U.S.R. Test The antigen used in this test is an alcoholic solution of 0.03 percent cardiolipin. 0.9 percent cholesterol and 0.21 i 0.0l percent lecithin. This standard \/.D.R.L. antigen and the standard buffered saline diluent for the preparation of the reagent V.D.R.L. antigen are commercially available.

Unheated Serum Reagin (U.S.R.) Test Preparation of U.S.R. Antigen-Dye Compound Reagent A. EDTA l0.l M)

Dissolve 3.72 g [(ethylenedinitrilo)tetraacetic acid disodium saltllEDTA) to a volume of l00 ml. B. Choline Chloride Solution percent) Dissolve the contents of a 250 g bottle of choline chloride in distilled water to a final volume of 625 ml. Filter and store at room temperature. C. Phosphate (0.02 M). Merthiolate (0.2 percent) Solution Dissolve 1.42 g Na HPO 1.36 g KH PO 1.00 g merthiolate in distilled water to a final volume of 500 ml. at a pH of 6.9.

Suspending Solution Solution A l part Solution 8 2 parts Solution C 4 parts Distilled Water l part Preparation of the Antigen Suspension 1.6 Ml of commercially available V.D.R.L. buffered saline are pipetted to the bottom ofa 30 m] flat-bottom. glass-stoppered vessel. Two (2.0) ml of antigen is added drop by drop so that it is added in about 6 seconds directly onto the saline solution. The vessel is then continuously but gently rotated on a flat surface while the antigen is being added. The last drop is blown out without touching the pipette to the saline solution. The vessel is rotated for an additional 10 seconds. Sixteen and four tenths 16.4) ml of standard buffered saline is then added and the vessel shaken (with the top on) bottom to top and back thirty (30) times in about 10 seconds. The resultant product is the V.D.R.L. antigen suspension used below.

The above prepared V.D.R.L. antigen suspension is centrifuged in an angle centrifuge at room temperature at a relative Centrifugal force of approximately 2.000 X g for minutes. The supernatant fluid is decanted by inverting the tube away from the side containing the sediment. The inside of the vessel is wiped with a cotton gauze without disturbing the sediment while the tube is held in an inverted position. The sediment is then resuspended in the above suspending solution with a volume equal to that of the original portion of antigen suspension that was centrifuged. If more than one container is used for centrifuging. the contents are pooled and mixed gently. The result is U.S.R. antigen. Seven hundredths (0.07) ml ofa 1 percent solution of Amethyst Violet", having the formula and identified as Basic Dye Cl. No. 50225, is slowly added to the U.S.R. antigen while the antigen is gently agitated. The result is an antigen preparation ready for testing.

Procedure for the Detection of Reagin Antibody (U.S.R. Test) The test may be conducted on serum or plasma samples which have not been heated. although heat inactivated samples are satisfactory.

Fifty p.l of serum and H60 ml of the above antigen preparation are added to a commercially available white test card. The mixture is briefly mixed with a mixing stick or other device. The card is then put on a clini EXAMPLE 2 Antigen Preparation To the V.D.R.L. antigen suspension (see above) is added 0.04 ml of 0.] percent alcoholic solutions of Nile Blue A, having the formula and designated as Basic Blue C.l. No. 51 180. The antigen preparation is now ready for use.

Procedure for the Detection of Reagin Antibody (V.D.R.L. Test) Plasma is removed from the blood cells of a suspect patient and heated at 56C. for 30 minutes. Fifty l of the sample and U60 ml of the above V.D.R.L. antigen preparation are placed on a commercially available white test card. After mixing, the card is placed on a clinical rotator and rotated as in Example 1. Aggregated dark blue particles visible under an incandescent. incident beam of light denote a positive syphilis test. A negative reaction is one in which the reagents do not aggregate into discrete particles but remain as a homogenous dispersion.

EXAMPLE 3 An antigen preparation is again produced as in Ex ample l. The test is conducted in the same manner as the U.S.R. test except that 1 pl of a 0.1 percent aqueous solution of polyvinylimidazoline is added to the test card and dried at room temperature before rotating. The aggregated particles are such that visibility thereof is further enhanced over the card wherein no imidazoline polymer is used.

EXAMPLE 4 and dried as in Example 3. Visibility of the aggregated particles is again amplified.

EXAMPLE 5 An aqueous solution of 0.1 percent of Azocarmine G". having the formula and designated as Acid Red-Cl No. 50085, is prepared and 0.5 ml. ofit are added to 1.0 ml of Reiter antigen. This constitutes the Reiter-dye compound anti gen preparation. One drop of this preparation is placed on a glass slide and one drop of suspect serum is added. followed by brief mixing with a wooden device. The resultant mixture is rotated for 4 minutes at 160 r.p.m. and read under a microscope at 2X power. Fluffy translucent particles of red intensity appear after standing 1 minute indicating a positive syphilis reactionv By Reitcr antigen, as used herein is meant that such as disclosed by De Alessandro et al. isolation and Purification of a Protein Antigen of the Reiter Treponema. Amer. Jour. of Syphilis and Gonorrhea. Vol 37, page 135 et seq. i953.

A typical testing procedure is set forth by Cammefax et all. Reiter Protein Complement Fixation Test for Syphilis, Public Health Reports, Vol, 72, page 335 et seq., I957.

Following the procedures of Examples l-S. various other dye compounds are substituted for the compounds used therein. The dye compounds correspond to Formula l, above. and, in each instance, show an easily detected, positive syphilis reaction via the presence of aggregated particles. The dye compounds used are set forth in Table l, below.

TABLE I l 1 Antigua and M COMPOUND z 1" EJONO. AR X Z Y AR Name and 6.1. NO- l I l i Q s 01 Cll 8351c am: at 0.1.

l NHCEH: No. 520m i H C l-lll Basic D- e 0.1. l i No. 511 5 l i s zlz -l l 0 C1 --cli2 i u i l s l 3 do 0 c1 figsiglllige 1o c.1. i l l l i N--N(Cll l l l I 9 5 do 0 Cl Basic Dye C.

r i No. 51195 i M a):

Oil 10 l 2 do 0 c1 l 3 512 1 2; 0.1.

i L] O 1 La];

11 4 do 0 C1 32512138 0.1.

12 l 3 Cl Basic Dye (3.1.

NO. 52000 HgN NH;

15 1 do S Cl Basic Dye [3.1.

No. 572cm; (CH3):

1% 2 S Cl Basic Dye 0.1.

No. 52010 ack NHCH;

15 2 do S Cl Basic Blue 0.1.

1 t M e): 520 5 Basic Green 5 C.I No. 62020 Basic Blue 17 0.1. No. 20 10 Basic Dye 6.1. No. 50050 Basic Dye C.I No. 500 10 Basic Dye 0.11. No. 500 45 Basic Blue 25 N( CH C.I. No. 52025 Basic Dye C.I. NHCZHS No. 50035 C1 Cal-l C1 CH 3 Tahic I C0nlinued USN] 647 02 mum L 6 5 C k C C C C C C e O 0 e 0 e e 5 e 0 J5 1 5 m 0; 1.6 8 m m my mm m mm c 5 c c 5 c m c 0 5 c c m) u m1 n u. 1 u n 1 D O a O- S a 0 O- 3 &N m C m N BN m m P N m N m m 2 H "mc N k z m C 0 m d n NH Q a H w 3 o o H o l 1 1 1 c c c m m C m m N N N o O d d o N N N d z 2 2 N 1 H c 2 C C a C 3 3 H 5 H H r\ H 2 2 4 v r L L CC) Q a v 6 55 I I4 I O I I t 2 02 o T m t C C C e 2O 10 C C C 0 CO C C 10 5 5 e5 e6 e0 e1 0N d d e u 0 e6 05 W1 W2 W2 i e E WE) '6 6 1 i 0 W 2 2 O V Ho Ho 2 W2 W2 B0 V0 2 2 O O O I N N O O 0 N N 0 0 c5 c5 c5 0 5 c G c5 c5 c5 c C c5 .1 1 1 1C5 1 .1 1 1 1 1 1 .1 M Mm nm a 5 S m a 8 a SI 8 S r 0 O o o N N m l C 0 MC MN & N w 5 m &N RN mN fic 3 um m 2 \M 2 H m h H c N "m Q m m I C N o HN NH 0 C N 0 d d HN m. a N 3 U H C o 0 0 0 o o o d C c m 3 H 1 1 l 1 l l l 1 l 1 1 1 C C C C C C C C C C C C N N N N N l \l S o H d C 3 o I|\ H K N 0 UN C N i N N C N N N 2 N 3 2 C N! 2 2 2 2 C N \J 2 H H a 2 \1 \J 1 3 2 H C c R C 2 H C C C 5 s C 2 C a 5 a H H 5 H 5 H H H H f\ m I\ I\ 2 2 q .l l i i Table i-Continued f I A N 61 Basic Red 6 f 0.1. No. 50375 i NH: i i 1, 5 14 01 d do Basic Red 6 0.1. No. 575 E HnN i 1 3 H N c1 Solvent Blue T i i Q c.z. No. 50 100 1 ii- 1 a i *7 N 01 do do Solvent Blue I; Q N C.I. No. 5ouoe 1 @ii 3 0 c1 Basic Dye C.I. I (113C)=N No. 51000 I I -9 H 0 c1 H Basic Blue 3 l (11 09 1! --N( u e 0 c1 NR Baaic Dye 0.1.

i i 51 do 0 C]. Basic Dye 0.1. 4 No. 51015 52 0 c1 Mordant Violet:

c.I. No. n w OH 51025 53 0 C1 Basic Blue 6 (30ml 0.1. No. 51175 no double chloride salt 55 a. 0. l-ZS percent by weight of a syphilis antigen rea- 1. An antigen preparation for use in carrying out an agglutination test for syphilis which comprises gent.

b. (10001-02 percent by weight of a dye compound comprising (H:C2)2N N= *N(C2H5)2 and i l c. a flocculating amount of polyvinylimidazoline. 2. The antigen preparation of claim 1 wherein said dye compound has the formula (W N (a -2):" N a -5) o= \NH2 3. The antigen preparation of claim I wherein said SO Na dye compound has the formula l I N on (H5c3)2N 0::

4. The antigen preparation of claim 1 wherein said 6. The method of claim 5 wherein said dye comdye compound has the formula pound has the formula 8. The method of claim wherein said dye compound has the formula 5 2): N- 2 c 5 SO NB.

N NH 7. The method of claim 5 wherein said dye comm pound has the formula "a (H 6 fl o m 9. A method for carrying out an agglutination test for syphilis which comprises mixing a test serum or plasma 01' with the antigen preparation of claim l. 

1. A TEST SERUM OR PLASMA AND
 1. AN ANTIGEN PREPARATION FOR USE IN CARRYING OUT AN AGGLUTINATION TEST FOR SYPHILIS WHICH COMPRISES A. 0.1-25 PERCENT BY WEIGHT OF A SYPHILIS ANTIGEN REAGENT, B. 0.0001-0.2 PERCENT BY WEIGHT OF A DYE COMPOUND COMPRISING
 2. AN ANTIGEN PREPARATION COMPRISING A. 0.1-25 PERCENT BY WEIGHT OF A SYPHILIS ANTIGEN REAGENT AND B. 0.0001-0.2 PERCENT BY WEIGHT OF A DYE COMPOUND COMPRISING
 2. The antigen preparation of claim 1 wherein said dye compound has the formula
 2. an antigen preparation comprising a. 0.1-25 percent by weight of a syphilis antigen reagent and b. 0.0001-0.2 percent by weight of a dye compound compriSing
 3. The antigen preparation of claim 1 wherein said dye compound has the formula
 4. The antigen preparation of claim 1 wherein said dye compound has the formula
 5. A METHOD FOR CARRYING OUT AN AGGLUTINATION TEST FOR SYPHILIS ON A TEST CARD HAVING DRIED THEREON A TEST SPOT CONTAINING A FLOCCULATING AMOUNT OF POLYVINYLIMIDAZOLINE SAID METHOD COMPRISING CONTACTING SAID TEST SPOT WITH
 6. The method of claim 5 wherein said dye compound has the formula
 7. The method of claim 5 wherein said dye compound has the formula
 8. The method of claim 5 wherein said dye compound has the formula
 9. A method for carrying out an agglutination test for syphilis which comprises mixing a test serum or plasma with the antigen preparation of claim
 1. 